Broadly neutralizing antibodies (bnAbs) are ideal components for preventing HIV infection. However, no vaccine candidate investigated so far has induced such holy grail components although selected individuals living with HIV have generated bnAbs after a 2 to 4-year delay. We hypothesize that a sequential induction pathway is involved in inducing bnAbs in these individuals, and propose new vaccination approaches to mimic the pathway by which HIV induces bnAbs as novel preventive HIV vaccine strategies. We will mimic this process by sequential immunizations with various structure-stabilized chimeric Env gp140 proteins (rEnvs). These rEnvs contain complete different strain-specific epitope profiles but share conserved weakly-immunogenic epitopes for bnAbs. Therefore they will not boost strain-specific immune responses but gradually boost bnAb responses against conserved determinants, mimicking the elicitation of bnAbs by the emerging new Envs in evading strain-specific antibody responses in HIV-infected individuals after long term infection, during which strain-specific epitopes undergo continuous antigenic variation. To increase immunogenicity, rEnvs will be constructed to include a trimerization sequence to stabilize their trimeric structures (cs-rEnvs). Highly immunogenic nanoparticles, designated as nanoclusters, will be self-assembled from these trimeric cs-rEnvs and used for immunization. Broadly reactive antibody responses will be evaluated in guinea pigs. Two specific aims will be pursued: 1) Generation and purification of different chimeric cs-rEnvs with conserved regions from the same Env but variable regions from Envs in different subtypes, and fabrication of nanoclusters assembled from the resulting cs-rEnvs; 2) Comparison of sequential-immunization regimens with nanoclusters self-assembled from different cs-rEnvs for induction of broadly reactive antibody, particularly bnAb responses, in guinea pigs by parallel intramuscular and intranasal routes. The long term goal of this proposal is to develop an effective HIV vaccine strategy to achieve preventive broadly neutralizing antibody responses. This project will provide proof of the concept that a sequential-immunization regimen with self-assembled nanoclusters containing different cs-rEnv trimers is a potential approach to develop a preventive HIV vaccine.